Quadrupole ion trap mass spectrometers (QITMS) are used to provide rapid and sensitive analyses of a wide range of chemical and biochemical compounds. QITMSs and related spectrometric methods are known in the art and are as provided in U.S. Pat. No. 4,540,884, the entirety of which is incorporated herein by reference. Such instruments have begun to play a particularly important role in proteomics research as their favorable characteristics are applied to the identification, quantitation, and structural elucidation of peptides and proteins. One limitation with the QITMS, however, is that the structural analyses provided by this instrument is performed in a serial manner in the context of tandem mass spectrometry (MS/MS) experiments. With the emerging importance of proteomics the need for more rapid analyses are being realized.
A range of ions with different mass-to-charge (m/z) values can be trapped simultaneously in a quadrupole ion trap by the application of a radio frequency (rf) voltage to the ring electrode of the device. The trapped ions all oscillate at frequencies that are dependent on their m/z, and these frequencies can be readily calculated. MS/MS is then performed by carrying out three steps. First, the analyte ions having the single m/z of interest (parent ions) are isolated by changing the rf voltage applied to the ring electrode and by applying waveforms (i.e. appropriate ac voltages to the endcap electrodes) with the appropriate frequencies that resonantly eject all the ions but the m/z of interest. Second, the isolated parent ions are then resonantly excited via the application of another waveform that corresponds to the oscillation frequency of the parent ions. In this way, the parent ions' kinetic energies are increased, and they undergo energetic collisions with the background gas (helium), which ultimately result in their dissociation into product ions. Third, these product ions are then detected with the usual mass analysis techniques in QITMS. It is the mass differences between these product ions and their incipient parent ions that provides the structural information during this MS/MS experiment. This method of performing MS/MS is the current state-of-the art in commercial QITMS, and referred to as serial MS/MS.
Multiplexed MS/MS refers to performing MS/MS on ions of multiple m/z ratios simultaneously. A primary concern, however, is that upon isolation and dissociation of several compounds simultaneously, the product ions that are formed need to be associated with the correct parent ions in order for structural information to be gathered for each parent ion. During serial MS/MS this is accomplished by isolating and dissociating only one parent ion at a time so that the resulting products necessarily come from that parent ion. When one isolates and dissociates multiple parent ions all at once, the normal manner of relating which product ions dissociate from each parent ion is lost.
Several protocols for multiplexed MS/MS on Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometers have been reported. Comprehensive 2-dimensional (2-D) methods analogous to 2-D NMR were used to simultaneously dissociate a collection of parent ions. Attributing the resulting product ions to the appropriate parent ions relies on a sinusoidal pattern of excitation waveforms that produces a modulation in the product ion abundances that can be later deconvoluted. Hadamard transform methods have also been used, but like the comprehensive 2-D approach multiple spectra are acquired in which different subsets of parent ions are simultaneously dissociated. The drawback to both the Hadamard method and the comprehensive 2-D approach is that these methods provide little to no timesavings as compared to the analogous serial approaches. Further, parent ion dissociations and product ion abundances do not always vary in the expected manner. Encoding is dependent upon known changes in parent ion kinetic energy, but product ion abundances do not necessarily change in a direct manner as parent ion kinetic energies (and, thus, collision energies) are changed. The net result is that product ions may be associated with incorrect parent ions.
Another approach developed recently allows product ion spectra to be obtained from multiple parent ions in a single mass spectrum, which significantly enhances the throughput. Because MS/MS analyses on FTICR mass spectrometers are inherently slower than MS/MS analyses on QITMS, this method is noticeably slower. Furthermore, this approach relies on the high mass accuracy of the FTICR to identify product ions from different parent ions by exact mass and database searching. Consequently, this method necessitates the high performance capabilities offered only by FTICR spectrometers, and therefore is not suitable for cheaper and more widely accessible mass spectrometers like QITMS. Further, this method depends upon the compound of interest present in an accessible data base and effective search capabilities—without which the analysis is unworkable.
Another QITMS limitation relates to the fact that externally-generated ions of different mass-to-charge (m/z) ratios are trapped with unequal efficiency after being transferred from the ion source. This unequal trapping efficiency results in mass spectra that do not accurately reflect the relative quantities of the analytes in a given sample. This m/z (or mass) bias arises because there exists, for each m/z ratio with a given kinetic energy, an optimum rf amplitude on the ion trap electrodes for efficient trapping. Furthermore, this optimum rf amplitude is different for each m/z ion. Such issues arise within or outside the context of MS/MS analyses.
To overcome mass bias, three different approaches have been used in the art. First, the rf amplitude can be increased in three different steps during ion accumulation. This step-wise increase results in optimum trapping of three different m/z ratios whether or not they exist in the sample. In effect this reduces the severity of the mass bias for a larger range of m/z ions, but does not totally eliminate it; the trapping efficiency for any give m/z ratio can still vary significantly. The step-wise increase in the rf amplitude cannot provide uniform trapping efficiency over a wide m/z range. The second approach, described in U.S. Pat. No. 5,729,014, involves a linear increase in the rf amplitude during ion injection. This linear increase in amplitude is meant to achieve, for a very brief time, the optimum rf amplitude for each ion in a given m/z range. While effective at reducing the mass bias associated with externally-injected ions, this method raises at least two issues—(1) increasing the rf amplitude in such a manner can lead to ion dissociation and thus loss of ion signal and sample integrity; (2) the linear rf amplitude increase (or multiple segments of this linear function) only approximates the non-linear increase necessary to match the ideally-predicted relationship.
A third approach has been developed in which the rf frequency is changed in the theoretically correct way. U.S. Pat. No. 6,121,610 describes a system in which the optimum rf frequency is varied in inverse proportion to the square root of the m/z ratio (i.e. (m/z)−1/2). Alternatively, there is described a method in which the rf amplitude is decreased during ion injection to accomplish an effect similar to that described by in the '014 patent. The methods of the '610 patent are theoretically preferred, but changing the rf frequency is very challenging from an electronics standpoint. Furthermore, ion losses for low m/z ions can still readily occur.